Pol. J. Environ. Stud. Vol. 26, No. 3 (2017), 1089-1096 DOI: 10.15244/pjoes/67534

Original Research Screening an effective aerobic strain and its performance in MBR

Jifeng Guo*, Ping Guo, Simeng Li, Xiaojie Li, Chongli Luo, Hui Wang, Qian Jiang

School of Environmental Science and Engineering, Key Laboratory of Subsurface Hydrology and Ecology in Arid Areas of Education Ministry, Chang'an University, Xi'an, 710064, P.R. China

Received: 6 June 2016 Accepted: 4 December 2016

Abstract

A highly effective aerobic denitrification strain was screened from the municipal activated sludge and its gene sequence was obtained from DNA extraction and amplified by the polymerase chain reaction (PCR) amplification. Based on the result, the strain was preliminarily identified to be by way of comparison with the National Centre for Biotechnology Information (NCBI) Genbank. In order to get more knowledge of the aerobic denitrification strain and its application in , the strain was added into the membrane bioreactor (MBR) to construct a denitrification MBR (dMBR) for municipal wastewater treatment due to its high denitrification ability. The experiment results showed that removal efficiency was as high as 80% in dMBR and nitrogen removal efficiency was 50% in the control MBR (cMBR), which indicated that aerobic denitrification in the MBR tank played a main role in removing the nitrogen under aerobic conditions. The best strain dosage was 20-30% in the total activated sludge. The dMBR also had a better membrane fouling resistant effect than cMBR. As a result, the dMBR could be an effective method for nitrogen removal of the wastewater treatment.

Keywords: screening, aerobic denitrification strain, membrane bioreactor (MBR), trans-membrane pressure (TMP)

Introduction [1-3]. In 1980s, researchers found aerobic denitrification strains from the effluent of the Due to the large amount of industrial wastewater denitrification treatment system, and they isolated aerobic discharge and long-term excessive use of agricultural denitrification strains such as Paracoccus pantotropha, fertilizers, nitrate water pollution is serious. Nitrate can Pseudomonas sp., and Alcaligenes faecalis, etc. [4-5]. be transformed into one of the three major carcinogenic Then more and more researchers investigated the aerobic nitrosamines, which are greatly harmful to human denitrification strains [6-8]. However, the studies had been health. Nitrogen is also a major substance that causes limited in strain screening and their culture conditions. It was seldom that the strains were put into the bioreactors for treating nitrogen wastewater. Membrane bioreactors (MBR) are activated sludge *e-mail: [email protected] processes where the secondary settling tank is substituted 1090 Guo J., et al. by a membrane filtration unit. The main advantages lie Enriched culture medium was composed of beef in the high sludge concentration in combination with an extract 1.0 g/L, peptone 5.0 g/L, and KNO3 1.0 g/L. excellent effluent quality [9-10]. b) Selective culture medium (DM culture medium) However, one disadvantage is that the MBR has Selective culture medium was composed of low denitrification because continuous aeration must be Na2HPO4•7H2O 7.9 g/L, KH2PO4 1.5 g/L, NH4Cl 0.3 g/L, performed to delay membrane fouling [11]. Despite the MgSO4•7H2O 0.1 g/L, sodium succinate 4.7 g/L, KNO3 existing simultaneous nitrification and denitrification 2.0 g/L, NaNO2 1.0 g/L, and pH 7-7.5. (SND) phenomena in MBR, the condition in the MLSS must be as high as 20~30 g/L [12] and the SND is also Domestication and pure strain separation limited. At the same time, the high MLSS concentration can result in serious membrane fouling. So far for The sample was inoculated in FM culture medium, most MBR designs, the traditional nitrogen removal then was cultured on the rotary shaker with intermittent method (adding anoxic (AN) tank) is adopted because aeration at 25ºC and 160 r/min for 5 d. Forty-eight types of its popularity in the treatment. For example, the pre- of strains were screened. Then these strains were ino- denitrification stage [13], the post-denitrification stage culated in DM culture medium at 25ºC and 160 r/min [14], and the sequencing batch membrane bioreactors [15] for 40 h. After that, measuring the total nitrogen (TN) are all used to remove nitrogen. These MBRs technologies concentrations in the bacterium suspension to select undoubtedly add to the investment. the highly effective strains by inspecting the bacterium An aerobic denitrification strain can remove nitrogen suspension’s TN removal ratio was above 50%. under aerobic conditions. If these strains were used in MBR [16], the above denitrification stage could be omitted Strains rescreening to save more investments. But there was competition for several kinds of aerobic denitrification strains in the long- The obtained strains were rescreened with simulated running of MBR, especially in the absence of a carbon municipal wastewater (Table 1) to get better denitrifica- source. And the aerobic denitrification strains might be tion performance strains. The strains were inoculated in degraded and lose their role of denitrification. So it is the above medium, and then were cultured on the rotary important that screening the aerobic denitrification strain shaker with intermittent aeration at 25ºC and 160 r/min could adapt to MBR’s running conditions (the absence of for 5 d. a carbon source). The better denitrification performance strains whose To get more knowledge of aerobic denitrification TN removal ratios were above 90% could be chosen. strains and its application in municipal wastewater, a kind of aerobic denitrification strain was screened from a local Enhanced strains rescreening wastewater treatment plant (A2/O process). The strain was applied in MBR to study the bioreactor’s performance The obtained strains were rescreened using supernatant of nitrogen removal, which is expected to be an effective liquor from an MBR treating simulated municipal way to solve the environmental issue of nitrogen removal. wastewater (almost without ammonia, but nitrate and ni- trite only, Table 2) to investigate the denitrification perfor- mance in order to enable the strains to get better adaption Material and methods in the actual operation of the sewage treatment process at 25ºC and pH 7, especially in the conditions of EPS and Strain screening SMP as the carbon source. Then the strain of the best nitrogen removal efficiency Microorganism’s origin will be chosen.

The sample came from the activated sludge in the Strains Identification aeration tank of the Fourth Municipal Wastewater Treatment Plant (MWTP) in Xi'an city, China. DNA of the screened strains were extracted and am- plified by polymerase chain reaction (PCR) amplification Culture medium method [18], then the samples were sent to Shanghai San- gon Biological Engineering Technology & Services Co., Culture mediums were prepared according to [17]. Ltd. for gene sequenceing. The bacterial morphology was a) Enriched culture medium (FM culture medium) observed by scanning electron microscopy (SEM).

Table 1. Simulated municipal wastewater. Total Nitrogen Ammonia Nitrate Nitrite COD Item Cr pH (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) Influent 45-50 20-45 2-20 0-3 270 6.9-7.2. Screening an effective aerobic... 1091

Table 2. Simulated municipal wastewater (without ammonia, but nitrate and nitrite only). Total Nitrogen Ammonia Nitrate Nitrite COD Item Cr pH (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) Influent 45-50 0-1 40-48 2-3 270 6.9-7.2

a) DNA extraction: sludge, which was named denitrification MBR (dMBR). Several single colonies from the culture medium The MBRs used to treat the municipal wastewater had a were picked up with inoculating loop and placed into total volume capacity of 1.0 L (Fig. 1). 200 mL Eppendorf pipes (with 30 μL sterile double- They were equipped with hollow-fiber ultra filtration distilled water), then heated at 94ºC in the water bath for (UF) membrane modules made of PVDF (Hangzhou 2-4 minutes so the cell wall would be broken and the inner Qiushi Membrane Technology Co., Ltd) with a total DNA would come out. Then the liquid could be as a DNA surface area of 0.015 m2 and a nominal pore size of template and ready to be amplified by PCR. 0.01 μm. The flux of the membrane was 12 L/m2·h. Air b) PCR amplification primer and procedure: was supplied through an axial perforated tube below the Sense primer P1: 27F (5’-AGAGTTTGATCCTGGC- membrane modules to supply demanded by the TCAG-3’), anti-sense primer P2: 1492R (5’-TACGGC- microorganisms and induce a cross-flow velocity along TACCTTGTTACGACTT-3’). The above primers were the membrane surface. The wastewater from the storage synthesized by Shanghai Invitrogen Biological Enginee- tank was supplied by an influent pump into the bioreactor. ring Technology & Services Co., Ltd., China. The HRT of the MBR was 6 hours. In the whole operation, PCR amplification procedure: the DNA template was the activated sludge was not discharged. The quality of the pre-denatured at 94ºC for 4 min, 30 cycles (denatured 45 s raw wastewater is listed in Table 1. The membrane-filtered at 94ºC, annealed 30 s at 60ºC, extented 90 s at 72ºC), and effluent was obtained by suction using a pump connected finally extented 12 min at 72ºC. The total volumes of PCR to the modules. The effluent flow rate and trans-membrane reaction system were 50 μL (sterile double-distilled water pressure (TMP) were monitored by a water meter and a 40 μL, 10×PCR reaction buffer 5 μL, 4×dNTP solution 1 vacuum gauge, respectively. μL, 10 mmol/L P1 1 μL, P2 1 μL, taq dna polymerase 1U, and DNA template 2 μL). Analytical items and methods DNA and PCR products were detected by 0.8% and

1% agarose gel electrophoresis, respectively (about Wastewater parameters, including CODCr, TN, and + 30 min, 150 V, observed under ultraviolet lamp), then sto- NH4 -N, were analyzed according to the Chinese NEPA red at -20ºC. FQ DL2000 DNA Marker was synthesized standard methods (1997) [19]. by Shanghai Biocolors Biological Engineering Technolo- gy & Services Co., Ltd. The DNA sequencing was intercompared by the Na- Results and Discussion tional Centre for Biotechnology Information (NCBI) Gen- bank of U.S.A. Strains screening and identification

Experiment setup According to the strains rescreening procedure, two kinds of aerobic denitrification strains whose TN removal Two MBRs were used in the experiment: one was only efficiency were both over 90% were screened: Strain 1 added with the activated sludge from the Fourth MWTP, and Strain 2. which was named controlled MBR (cMBR); another was added with aerobic denitrification strains and the activated

Fig. 2. PCR amplification. 1: Marker, 2: Negative Control, 3-1: PCR product of Strain 1, Fig. 1. Membrane bioreactor (MBR) sketch. 3-2: PCR product of Strain 2 1092 Guo J., et al.

Table 3. Sequence length and closest phylogenetic affiliation of two strains.

Sequence Phylogenetic relationship Strains length Species Similarity 1 190 Pseudomonas sp. 193/195(98.9%) 2 168 paracoccus sp. 209/219 (95.4%)

the phylogenetic tree of the denitrification strain, which was constructed using molecular evolutionary genetics analysis (MEGA 6) software [20] according to the neighbor-joining method [21]. It was confirmed that Pseudomonas bacteria genera was common [22]. As shown in Fig. 4, Pseudomonas sp. strain W10 16S ribosomal RNA gene partial sequence, Uncultured Pseudomonas sp. 16S Fig. 3. SEM photos of strains. rRNA gene clone 65p38, Uncultured Pseudomonas sp. clone 361 16S ribosomal RNA gene partial sequence, and Pseudomonas sp. Isoamylase gene complete cds were The extracted DNA was PCR-amplified by amplified relatively close in genetic distances. These four kinds of primers. Observed by agarose gel electrophoresis, the Pseudomonas sp. strains had different genetic relationships amplified fragments were both about 1,500 bp (shown in from Pseudomonas fluorescens Pf0-1 and Pseudomonas Fig. 2), and were proved to be target fragments without sp. 2 - haloalkanoic acid dehalogenase genes. The latter tails. The results of PCR amplification were satisfied. two kinds of strains belonged to the same branch, but their similarity was smaller. Characteristics of the strains Species-rich Both strain cells had short-rod shapes and were gram negative. And their colonies were white with circle points A 1 mL sample was inoculated in enriched culture in the middle and smooth edges. The strains’ shapes are medium (FM) contained in a 1,000 mL conical flask, then shown in Fig. 3 (SEM photos). was cultured on a rotary shaker at 25ºC and 160 r/min for 5 d. Then all the bacteria suspensions were filtered by filter paper. The intercepted strains were stored for use. After Enhanced strain rescreening species-rich, the net weight of strains in 1,000 mL conical flasks was 0.5g. The two kinds of strains (1 and 2) that had good denitrification performance were selected by the Dosage of strain enhanced rescreening method. The results showed that the TN removal efficiency of strains 1 and 2 were 50% Activated sludge MLSS in the reactor is 8 g/L. In and 10%, respectively. Then strain 1 was selected as the order to maintain the same amount of sludge amount, experimental strain. some activated sludge in the dMBR should be discarded. The gene sequences from the bands were sequenced by For example, for the dosage of 10% of the bacteria in Sagon Company (Shanghai, China) and were compared the dMBR, two 1,000 ml conical flasks with aerobic with the NCBI Genbank. And the strains were preliminarily denitrification strains were needed, while removing 1 g identified to be Pseudomonas, whose coverage was activated sludge from the dMBR. The procedure is: 1 L 98.9% with the target sequence (Table 3). Fig. 4 showed of activated sludge in the reactor was centrifuged at

Fig. 4. The phylogenetic tree of the strain. Screening an effective aerobic... 1093

3,000 rpm for 10 minutes, and 200 mL supernatant was and 50% dosage of strains were added to the dMBR, discarded to become 800 mL volume. A 1 L conical respectively, while reducing the amount of activated flask with species was also centrifuged at 3,000 rpm for sludge to enable the total MLSS concentration in dMBR 10 minutes, and 900 mL supernatant was discarded to of 8 g/L. Runtime of the two MBRs was 10 days after become 100 mL volume. Two bottles of this 100 mL every dosing to investigate the removal efficiency of the concentrated strain and 800 mL of concentrated activated pollutants – especially nitrogen removal efficiency. sludge were mixed, and 10% of the strain dosage was obtained. CODCr removal

Performance of two MBRs As can be seen in Fig. 5, CODCr removal was significant in the two reactors. There was no great influence on the

It must be asserted that the influent was well buffered early removal of CODCr due to the MBR’s characteristics and accordingly the reactor pH throughout phases of of the high sludge concentration. Of course, the sludge operation was between 6.8 and 7.4. The ammonia stripping was taken from the municipal wastewater treatment could be neglected because ammonia in water with such a plant aeration tank, which could be well adapted to the range of pH existed mostly as ammonium ions at ambient simulated municipal sewage. temperature. Of course assimilation was responsible for In the whole stage, the CODCr concentration of 25~30% of the nitrogen loss in the biological wastewater effluent fluctuated with the influent and was relatively treatment process [23]. The endogenous respiration could stable. Then at the later of every stage, activated sludge also compensate a lot for the nitrogen loss in the MBR in cMBR became mature and the EPS in the bioreactor with long SRT 20~40 d [24]. Therefore, the dissimilation was produced more and more, so the CODCr of by bacteria mainly contributed to nitrogen loss in the supernatant liquor rose gradually (Fig. 5a) [25]. But the ordinary MBR and traditional nitrogen removal (TNR), CODCr concentration of effluent was still steady due to or SND would take the main contribution in nitrogen membrane interception (Fig. 5b) [26]. At the same time, removal. dMBR showed slightly different CODCr concentrations In fact, two parallel MBRs were operated on under in supernatant liquor from the cMBR, that is the dMBR’s the same conditions (Intermittent filtration: 12 min supernatant liquor CODCr concentration was reduced filtration and 3-min pause; DO is 3-4 mg/L; MLSS is 8 gradually, and the CODCr removal efficiency was 83%

g/L) to investigate the relationship of dosage of strain when the CODCr concentration of influent fluctuated at and pollution removal efficiency. 10%, 20%, 30%, 40%, 270 mg/L. The aerobic denitrification bacteria strain

Fig. 5. Variations of CODCr removal of the MBRs. a) Supernatant COD variations of two MBRs, b) Effluent COD variations of two MBRs. 1094 Guo J., et al.

Fig. 6. Variations of ammonia removal.

1 in dMBR could adapt to the situation of lacking carbon bacteria did not have the function to make the ammonia source and take the CODCr in the supernatant as foodstuff converted to nitrate. The reason was that there was not to go on denitrification. So in spite of the whole sludge an anoxic environment for the denitrification bacteria. being in the respiration phase [27], there still was CODCr Also, in the first stage the TN removal in cMBR was not consumption in the dMBR supernatant. distinct because the denitrification needed a rather longer procedure than in nitrification. With the development of Ammonia removal the cMBR, the denitrification bacteria of the activated sludge and the TN removal was better than before. The As shown in Fig. 6, ammonia removal was high highest TN removal ratios were above 50%. in both of the MBRs, which indicated that ammonia- With different strain dosages, different changes took oxidizing bacteria played a role in removing the ammonia. place in the dMBR. From Fig. 7 we could see 10%, 20%, The ammonia-oxidizing bacteria and nitrobacteria of 30%, 40%, and 50% of the dosage to the whole activated the activated sludge were intercepted in the activated sludge, and 60%, 74.5%, 79%, and 80% TN removal, + sludge system by membrane, and the NH4 -N removal respectively. The results indicated that the aerobic ratio was over 99%. The advantage of MBR, providing denitrification bacteria played a role in denitrification and better retention of slow-growing microorganisms (like the TN removal may have increased with the increasing + nitrobacteria) [28], enhanced NH4 -N removal. strain dosage, which remained at about 80% TN removal under 30-50% dosage. TN removal DO is particularly important regarding ecological indicators for the aerobic denitrification bacteria. Patureau As shown in Fig. 7, TN removal in the dMBR was et al. [29] studied the aerobic denitrification strain more satisfying than that in the cMBRs. No doubt, the Microvirgulaaero, and they drew the conclusion that it nitrogen removal benefited from the strain in the MBR had no influence on the strains’ denitrification efficiency tank. In the cMBR, it was not a similar experience in when DO concentrations were below 4.5 mg/L, as ammonia removal, indicating that the denitrification denitrification activity began to rise sharply and

Fig. 7. Variations of total nitrogen removal. Screening an effective aerobic... 1095

Conclusions

1) A kind of high denitrification ability aerobic denitrification strain was screened, which was preliminarily identified as Pseudomonas. And the strain had the ability to use part of EPS and SMP in the MBR supernatant as a carbon resource. 2) The strain was put into the MBR to make a dMBR for municipal wastewater treatment. The TN removal efficiency was as high as 80% in dMBR, and the TN removal efficiency was 50% in the control MBR (cMBR). The aerobic denitrification strain in the MBR tank played a main role in removing nitrogen Fig. 8. Variations of TMP in the two MBRs with operation time. under aerobic conditions. The best strain dosage was 20-30% with total activated sludge. 3) The dMBR had a better membrane fouling resistant denitrification rates increased greatly. In the MBR tank, effect from the TMP variations in the two MBRs. although continuous aeration must be given to defect the membrane, DO concentration usually was below 4.5 mg/L due to the high MLSS concentration. And the ordinary Acknowledgements denitrification bacteria could be inhibited under the MBR’s DO conditions. dMBR provided a more suitable This study was financially supported by the Shaanxi environment for aerobic denitrification bacteria to compete Natural Science Fund (2014JM7256), the Shaanxi against other bacteria [12], and good nitrogen removal Construction Department Science and Technology (project could be obtained by aerobic denitrification bacteria. No. 2014-K28), the Special Transport Technological C/N ratio is also a very important factor. Previous Fund for Western China (2012318361110), the National studies [30-31] researched the effect of carbon sources on Natural Science Foundation of China (Nos. 51108437 SND and the results were always the same – that a higher and 21407012), the Chang'an University Students C/N ratio can achieve better SND efficiency. In this study, Innovation Program (201510710073), and the Special since the aim was to simulate the actual sewage treatment, Fund for Basic Scientific Research of Central Colleges, the wastewater used in experiments was adjusted to real Chang'an University (CHD2009JC057, 310829172002, domestic wastewater, the C/N ratio ranged from 6 to 8 310829163406). The authors are very grateful to Dr. all the time. In the latter part of the operation, the carbon Yaqian Zhao from University College Dublin and to Dr. source had changed (MBR will produce some substances Kun Lu from Stanford University for their editing. such as EPS and SMP in supernatant, which could be s carbon source for Strain 1); good nitrogen removal was achieved with the strain dosage increasing in the dMBR References and was not obtained in the cMBR. However, because of the difficult degradation of EPS and lack of other carbon 1. MATTHEW. P. HOCH, KEVIN S. DILLON, RICHARD sources, it was not easy to achieve better nitrogen removal. B. COFFIN, LUIS A. CIFUENTES. Sensitivity of bacterioplankton nitrogen metabolism to eutrophication in Thus, 20-30% of the dosage is appropriate. sub-tropical coastal waters of Key West, Florida. Marine Pollution Bulletin. 56 (5), 913, 2007. Membrane fouling 2. DOMAGALSKI J., LIN CH., LUO Y., KANG J., WANG S., BROWN L.R., MUNN M.D. Eutrophication study at Membrane fouling was investigated by the TMP the Panjiakou-Daheiting Reservoir system, northern Hebei changes. Fig. 8 showed the variations of TMP of the two Province, People›s Republic of China: Chlorophyll-a model MBRs. As shown clearly in the figure (especially after and sources of phosphorus and nitrogen. Agricultural Water 50 days operation), the TMP increased more slowly in Management. 94 (1-3), 43, 2007. the dMBR than in cMBR in the experimental period, 3. DJAMBAZOV G., PERICLEOUS K. Modelled atmospheric contribution to nitrogen eutrophication in the indicating that the dMBR has a high ability to tolerate English Channel and the southern North Sea. Atmospheric the membrane fouling compared with the cMBR. The Environment. 102, 191, 2015. denitrification strain in the dMBR might play a great role 4. ZHANG P., ZHOU Q. Simultaneous nitrification and in the resistance of the membrane fouling because the denitrification in activated sludge system under low oxygen strain in dMBR could adapt to the situation of lacking concentration. Frontiers of Environmental Science & Engineering in China. 1 (1), 49, 2007. carbon source and take the CODCr in the supernatant as foodstuff to go on denitrification, which could reduce the 5. PAI S.L., CHONG N.M., CHEN C.H. Potential membrane filtration pressure. applications of aerobic denitrifiying bacteria as bioagents in wastewater treatment. Bioresource Technology. 68 (2), 179, 1999. 1096 Guo J., et al.

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